There is considerable interest in using skeletal muscle progenitor cells, or satellite cells, for cellular therapies and tissue engineering applications. These mononuclear satellite cells, known in the context of skeletal muscle as myoblasts, must fuse into multinucleated myotubes in a highly coordinated differentiation process. Like native muscle, an engineered construct should produce an aligned bundle of myotubes to produce mature muscle ultrastructure that functions properly. Different stimuli, such as mechanical stretch, have been shown to enhance this process. Although mechanical stimulation can promote differentiation, mature muscle fibers have not yet been produced in vitro. Recently, microRNAs (miRNAs) have been shown to regulate gene function to influence proliferation or differentiation in skeletal muscle. As such, miRNA delivery to mechanically stimulated myoblasts may represent an important and novel way to enhance differentiation. The proposed research will examine miRNA expression in skeletal myoblast proliferation and differentiation in response to different mechanical stimuli. In our preliminary data we have identified some novel stretch-sensitive microRNAs and show that the specific stretch regimen produce statistically significant changes in miRNA levels. We will test the following hypotheses: (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via different sets of miRNAs that are temporally regulated, (2) stretch-sensitive miRNAs affect key proteins involved in myoblast differentiation and (3) differentiation can be enhanced by a combination of mechanical stimulation and regulation of miRNA levels. The specific aims of the proposed research are: (1) identify stretch-induced miRNAs by assessing the temporal effect of different levels of stretch, frequency and rest period on miRNA expression in myoblasts, (2) identify key downstream targets and functional effects of miRNAs during the response to mechanical stimulation, and (3) modulate miRNA levels to enhance the effect of applied stretch on myotube formation and differentiation in two-dimensional skeletal muscle cell cultures. Completion of these aims will provide important insights into the mechanism by which stretch mediates differentiation and new tools to modulate skeletal muscle function for applications in regenerative medicine. PUBLIC HEALTH RELEVANCE: This research involves a novel approach to promote the differentiation of cells into tissues using microRNAs, which have been recently identified as regulators of tissue development, and differentiation. While the study focuses upon the demonstration of proof-of-principle in skeletal myoblasts, the results can be extended to other tissues. The work, if successful, could have wide applicability both in the development of engineered tissues and cellular therapies.